Spread of Tfam isoforms in primate species

Tfam is a single copy nuclear gene structured in seven exons and six introns coding for the mitochondrial transcription factor A. Tfam is a member of the HMG (high-mobility-group) protein family, having two HMG boxes. It enhances mtDNA transcription by mitochondrial RNA polymerase in the presence of mitochondrial transcription factor B and is essential for mtDNA maintenance. ft is known the presence of a smaller isoform of the Tfam gene originated by an alternative splicing mechanism of the exon 5 has been identified in human and rat constituting respectively about 30% and 10% of Tfam transcripts in ali tissues. Moreover the presence of a processed pseudogenes lacking exon 4 of the gene has been recently reported in Presbytis cristata. This one could be the trace of a now extinct alternative transcript of Tfam gene. Here we present the search of alternative splicing isoforms of Tfam gene in different Primate's species through several approaches. Materials and Methods: Lymphoblastoid (Pongo pyg-maeus, Pan troglodytes, Papio anubis, Hylobates lar, Pan paniscus, Presbytis cristata) and fibroblastoid (Lemur catta) celi lines were used. Total RNAs were reverse tran-scribed wíth the Super Script TM III One-Step RT PCR using a standard protocol. For radioactive experiments a 3Z P dCTP has been used. RESULTS: RT PCR experiments were carried out using radioactive nucleotides and primers designed on exons 2 and 7 of the gene revealing the presence of the Tfam 05 isoform only in hominids. In Presbytis cristata a standard RT PCR experiment with the same pair of primers was performed. The products were differentially hybridizated with two different probes separately, one corresponding to exon 4 and the other to exon 5. The result of these hybridizations shows only the presence of the band relating to the Tfam complete transcript. RT PCR experi-ments, using a pair of primers designed on exons 1 and 5 of Tfam gene has revealed the presence of a splicing isoform containing a tract of intron I of the gene. We have analyzed the three open reading frames of the new ísoformobserving several stop codons and so excluding that this isoform is translated